Inhibition of abnormal cell growth with corticotropin-releasing hormone analogs

ABSTRACT

The glutamic acid residue of corticotropin-releasing hormone analogs have had the position 20 amino acid residue replaced with a D-amino acid moiety. The resulting CRH analogs do not significantly lower blood pressure but have anti-proliferative actions in cell culture and inhibit experimental cancer growth in animals (mice and rats). Novel applications of such analogs are described, such as to inhibit abnormal cell proliferation for conditions such as cancer, including melanoma, and for inflammatory dermatoses, such as psoriasis.

FIELD OF THE INVENTION

[0001] The invention generally relates to treatments for abnormal cellproliferation, particularly for treating epidermal disorders, and moreparticularly relates to a method of inhibiting abnormal cell growth withthe use of certain corticotropin-releasing hormone (“CRH”) analogs.Inhibition of abnormal cell growth has therapeutic utility in conditionssuch as cancer and psoriasis.

BACKGROUND OF THE INVENTION

[0002] Corticotropin-releasing hormone (CRH, also called CRF orcorticoliberin) was first characterized as a 41-residue peptide isolatedfrom ovine hypothalami by Vale et al. (1981). Subsequently, the sequenceof human-CRH was deduced from cDNA studies and shown to be identical torat-CRH, and then caprine, bovine, porcine, and white sucker fish CRHwere characterized. The CRH of hoofed animals show considerabledifferences from man, but the pig and fish sequences differ from thehuman/rat sequence by only 2 out of 41 residues.

[0003] For some mysterious reason, peptides with homologous structuresto mammalian CRH are found in cells of certain frog skins and in theurophysis of fish. In fact, the structure of sauvagine, the 40-aminoacid peptide isolated from the skins of Phyllomedusa frogs, was reportedseveral years before Vale's description of ovine-CRH. The structure ofsucker fish urotensin I was reported just months after the descriptionof ovine-CRH and resulted from an independent line of inquiry byLederis's group in Canada. Although sauvagine and urotensin I releaseadrenocorticotropin (ACTH) from the pituitary, the functions of thesepeptides in the tree-frog (Phyllomedusa species that live in aridregions of South America) and in the sucker fish remain unknown. TABLE 1Peptides of the Corticotropin-Releasing Hormone Superfamily SEQ. ID NO.PEPTIDE SPECIES SEQUENCE^(a, b)  1 CRH Human/ SEEPPISLDL TFHLLREVLEMARAEQLAQQ AHSNRKLMEII rat  2 CRH Pig SEEPPISLDL TFHLLREVLE MARAEQLAQQAHSNRKLMENF  3 CRH Sucker SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKMMEIFfish  4 CRH Sheep/ SQEPPISLDL TFHLLREVLE MTKADQLAQQ AHSNRKLLDIA goat  5CRH Cow SQEPPISLDL TFHLLREVLE MTKADQLAQQ AHNNRKLLDIA  6 Urotensin ISucker NDDPPISIDL TFHLLRNMIE MARIENEREQ AGLNRKYLDEV fish  7 Urotensin ICarp NDDPPISIDL TFHLLRNMIE MARIENEREQ AGLNRKYLDEV  8 Urotensin I MaggySEEPPMSIDL TFHMLRNMIH RAKMEGEREQ ALINRNLLDEV sole  9 Urotensin IEuropean SEDPPMSIDL TFHMLRNMIH MAKMEGEREQ AQINRNLLDEV flounder 10Urocortin Rat DDPPLSIDL TFHLLRTLLE LARTQSQRER AEQNRIIFDSV 11 UrocortinHuman DNPSLSIDL TFHLLRTLLE LARTQSQRER AEQNRIIFDSV 12 SauvagineFrog >EGPPISIDL SLELLRKMIE IEKQEKEKQQ AANNRLLLDTI

[0004] In humans CRH regulates, via release of proopiomelanocortin, ACTHsecretion from the anterior pituitary and has several direct actions oncentral and peripheral tissues. CRH has also been found to have directanti-inflammatory properties. More recently, evidence has been providedthat mammalian skin cells both produce CRH and express functional CRHreceptors (Slominski et al., FEBS Lett., 374, pp. 113-116, 1995;Slominski et al., J. Clin. Endocrinol. Metab., 83, pp. 1020-1024, 1998;Slominski et al., Hum. Pathol., 30, pp. 208-215, 1999), although it wasnot known whether locally produced CRH had an additional role in thephysiology of the skin, other than as a vasodilator and inhibitor ofthermal injury-induced edema.

[0005] Some therapeutic methods and uses for CRH are described byinventors Wei and co-workers in U.S. Pat. No. 4,801,612, issued Jan. 31,1989, titled “Method of Inhibiting Inflammatory Response,” and U.S. Pat.No. 5,137,871, issued Apr. 26, 1994, titled “Treatment to Reduce Edemafor Brain and Musculature Injury.” These patents describe the use of CRHto decrease the leakage of blood components into tissues produced byvarious adverse medical conditions, and thus to treat a patient forinjury to or disease of the brain, central nervous system or musculaturein which edema is a factor.

[0006] U.S. Pat. No. 5,869,450, issued Feb. 9, 1999, inventors Wei etal., describes CRH analogs in which the fifth amino acid from theN-terminus is D-Pro or in the case of urocortin or sauvagine where thefourth amino acid from the N-terminus is D-Pro or D-Ser. These analogshave an anti-inflammatory activity and a disassociated ACTH response.

[0007] Cyclic CRH agonists have recently been described by Rivier et al.(U.S. Pat. Nos. 5,844,074 and 5,824,771). These CRH analogs, modified bycyclization of residues 30-33 of CRH via a glutamic acid-lysine bridge,are more potent than native CRH in the release of ACTH and have lowermolecular weight than native CRH. The elimination of residues 1-3 or1-11 at the N-terminus of CRH has been shown to not alter biologicalactivities or ACTH-release potency. (Kornreich et al., J. Med. Chem.,35, pp. 1870-1876, 1992; Koerber et al., J. Med. Chem., 41(25), pp.5002-5011, 1998.)

[0008] Recently, Tjuvajev et al. (Tjuvajev, J., Kolesnikov, Y., Joshi,R., Sherinski, J., Koutcher, L., Zhou, Y., Matei, C., Koutcher, J.,Kreek, M. J., and Blasberg, R. Anti-neoplastic properties of humancorticotropin releasing factor: involvement of the nitric oxide pathway.In Vivo., 12, pp. 1-10, 1998. Department of Neurology, Memorial SloanKettering Cancer Center, New York, N.Y. 10021, USA) have introduced yetanother novel mechanism for CRH in the form of anti-cancer action.Tjuvajev et al. (1998) reported a series of in vivo and in vitro studiesthat evaluated the anti-neoplastic potential of CRH in W256 rat mammarycarcinoma. Using magnetic resonance imaging (MRI) and directmeasurements of tumor and peritumoral brain water content they foundthat CRH treatment (100 micrograms/kg subcutaneously twice a day for 3days) caused significant inhibition of growth ofintracerebrally-injected W256 tumor cells. CRH also exhibitedantiproliferative effects in in vitro cultures of W256 cells. Theantiproliferative effects of CRH in W256 cells involve activation ofnitric oxide synthase (NOS) and L-arginine-NO pathways. CRH activatedthe release of NO in W256 cells. The NO then became cytotoxic to thecancer cells.

[0009] Human trials of CRH for the treatment of peritumoral brain edemahave been initiated and preliminary data indicated that CRH reducedbrain edema associated with tumor metastases. However, the limitingfactor on the use of CRH has been the known blood-pressure loweringproperty of CRH. CRH causes relaxation of smooth muscles surroundingblood vessels and causes vasodilation, resulting in a lowering ofblood-pressure. The hypotension so produced is sufficiently dangerous tolimit the dosages of CRH that can be administered to humans. If thisdose-limiting toxicity is overcome by improved molecular design of CRHsuperfamily molecules, then it is conceivable that these analogs willhave a higher therapeutic index and have utility via theanti-proliferative mechanism of action.

SUMMARY OF THE INVENTION

[0010] In one aspect of the present invention, a method of inhibitingabnormal cell proliferation in a patient diagnosed as being at risk ofsuch condition is provided. In another aspect, practice of the inventionprovides a method of treating excessive epidermal proliferation. In yetanother aspect of this invention, a kit is provided that is useful fortreating a patient suffering from an abnormal cell proliferationconditions. The kit includes a pharmaceutically acceptable formulationof a corticotropin-releasing hormone (“CRH”) analog where the analog hasa D-configuration amino acid residue at a particular location of thesequence, and the kit further includes instructional materials fortherapeutic use of the pharmaceutically formulated peptide.

[0011] These inventive aspects relate to our having found thatreplacement of the glutamic acid residue at position 20 of CRH to aD-amino acid moiety creates a molecule that has minimal activity tolower blood pressure, but the molecule retains anti-proliferativeactions in cell culture and inhibits experimental cancer growth inanimals (mice and rats).

[0012] Thus, novel applications of [D-Xaa²⁰]-substituted analogs of CRH,such as a preferred embodiment [D-Glu²⁰], are herein described in whichthe ability to inhibit abnormal cell proliferation with reducedhypotension is utilized. The peptide analogs, such as are exemplified by[D-Glu²⁰] CRH, have amino acid sequences of the CRH superfamily butwherein at least one amino acid residue at the 20 position has beenreplaced with a D-amino acid residue (however, peptides having 40 aminoacid residues have the nineteenth such residue from the N-terminusmodified by inclusion of a D-amino acid).

[0013] Among the novel uses of these peptide analogs are for thetreatment of cancer and psoriasis, yet while avoiding the hypotensiveproperties of the normal CRH.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 graphically illustrates the hypotensive effects ofunmodified CRH by contrast to the effect of [D-Glu²⁰]-CRH inanesthetized male rats; and,

[0015]FIG. 2 graphically illustrates inhibition of cell proliferation byadministrations of a preferred embodiment in accordance with theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention describes novel actions of and applicationsfor CRH compounds that are analogs of the CRH superfamily of peptides.By “CRH superfamily” is meant to include those peptides recognized bythe art as belonging to the CRH family due to many sequence similaritiesand similar biological activities. These include the peptidesillustrated by Table 1. Thus, the CRH superfamily includes the CRHpeptides originating with or derived from a number of species, e.g.,rat, human, pig, sheep, cow, and fish, and also includes sauvagine,urotensin I, and most recently urocortin. Urocortin is a mammalianneuropeptide described by Vaughan et al., Nature, 378, pp. 287-292(1995).

[0017] The CRH peptides of this invention are based upon our discoverythat replacing the twentieth amino acid (in the case of those 41 aminoacid residue containing peptides of this family) with a D-amino acidreduces blood-pressure lowering yet while providing anti-neoplastic andanti-cell proliferative activities. Similarly, replacing the 19thresidue of peptides having 40 amino acid residues with a D-amino acidresidue also reduces blood pressure lowering yet providesanti-neoplastic and anti-cell proliferative activities.

[0018] We believe that the chirality of amino acid residue 20 is a moreimportant determinant of activity (such as at the CRH₂β receptor) thanthe acidic function (of the normal 20 position glutamic acid) or thanthe longer aliphatic side chain (or skeleton) of the glutamic acidresidue. In other words, we believe that the 20^(th) position is highlyspecific and that the recognition site does not particularly distinguishbetween amino acid residue structures, but instead primarilydistinguishes between chiralities. Accordingly, we believe thatsubstantially all peptides of this invention that have a D- orientationon the α-carbon of the amino acid residue at position 20 are suitable.

[0019] Also, among the CRH analogs described in the art are thoseincluding cyclic bonds, such as between the residues in the 30 and 33position, which may be a disulfide linkage (between two Cys residues)but preferably where each is an amide-bond (i.e., a lactam bridge).Suitable such cyclic analogs of the CRH family of peptides are thosedescribed by U.S. Pat. No. 5,844,074, issued Dec. 1, 1998, inventorRivier, which is incorporated herein by reference. Uses of such cyclicanalogs are suitable in practicing the subject invention, if such cyclicanalogs also have a D-amino acid substitution at the 20th amino acid (inthe case of those 41 amino acid residue containing the peptides of thefamily). Two examples of such suitable cyclic peptides are:Cyclo(30-33)-Acetyl-Pro⁴, D-Phe¹², D-Glu²⁰, Nle^(21,38), Glu³⁰, D-His³²,Lys³³-hCRH(4-41); and Cyclo(30-33)-Acetyl-Pro⁴, D-Phe¹², D-Glu²⁰,Nle^(18,21), Glu³⁰, D-Ala³², Lys³³-carp urotension (4-41).

[0020] The suitable neuropeptides should be administered under theguidance of a physician, and kits for practicing the invention arecontemplated which include instructional materials. When treatingdisorders on the surface of the skin, the administration is preferablytopical. Administration can be intermittent or continued after diagnosisof the premalignant, neoplastic or psoriatic condition, until there isalleviation of symptoms or signs of the diseases.

[0021] One aspect of the present invention is to provide a kit in whicha pharmaceutically suitable formulation of the peptide (that is, the CRHanalog) is one component of the kit while accompanying the peptide areinstructional materials for therapeutic use of the so-formulatedpeptide. Instruction includes mode and manner of administration,dosages, frequency, contra Indications, and the like. For example, theinstructional materials of such a kit instruct the patient in theappropriate dose and regime for administering the peptide.Therapeutically effective dosages are discussed hereinafter.

[0022] While the instructional materials typically comprise written orprinted materials, they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto magnetic storage media (e.g., magnetic discs, tapes, cartridges,etc.) electronic storage media (e.g., memory chips, processing chips,etc.) optical media (e.g., holographic media, CD ROM, etc.), and thelike. Such media may include addresses and/or hotlinks to globalelectronic access sites that provide such instructional materials.

[0023] Because peptides of this invention have the property ofinhibiting abnormal cell proliferation, they are useful in a number ofdifferent therapeutic applications. Specific tissues for which clinicalusage of these peptides may be applied include skin, as well as itsadnexal structures such as hair follicle and sebaceous glands, and otherepithelial tissues (eyelids, nasal membranes, oropharyngeal membranes,upper respiratory tract, esophagus, lower digestive tract), skeletalmuscle, smooth muscle, cardiac muscle, blood vessels of the brain, andblood vessels of the lungs and kidneys. Where the tissues are notreadily reached by topical administration (such as by creams and thelike as further described hereinafter), then parenteral or systemicadministration may be used.

[0024] For example, therapeutic uses of these peptides includeadministration to treat disseminated cancer, including melanoma,squamous cell carcinoma, breast cancer, premalignant lesions such aslentigo maligna, actinic keratosis, and, for non-cancerous conditions,such as psoriasis, eczema, alopecia areata, hypertrichosis or keloids.The keratinocytes are cells that line the base of the epidermis and formnew cells which cover the surface of the body. These cells have a highmetabolic activity and turnover; moreover, they participate in theinflammatory response, as they actively secrete cytokines and attractother inflammatory cells from the body (white blood cells). A disruptionof keratinocyte activity is prominent in inflammatory dermatoses, ofwhich psoriasis is a primary example. Other related conditions areeczema and various forms of dermatitis. Thus, an agent which inhibitskeratinocyte proliferation is an useful agent for therapy ofinflammatory dermatoses. For example, the basic lesion in psoriasis ishyperproliferation of keratinocytes in the epidermis. The turnover rateof these cells may be ten times more rapid than usual, and maturation ofthe cells is abnormal. (J. H. Stein, editor, Internal Medicine, chapter216, “Psoriasis,” pp. 1300-1302, 1998.)

[0025] The hair follicle—sebaceous gland unit of the skin, part of the“adnexa” or appendages of the skin is of pharmacological interest forthese reasons:

[0026] (a) a large peptide such CRH, with a molecular weight of 4754daltons, can get to targets because it can be formulated to sit on theskin and penetrate along the hair shaft to the base of the hair follicleand to the sebaceous gland;

[0027] (b) proliferation of hair follicle cells can result inhypertrichosis, so a peptide like CRH may have value as a means forstopping excessive hair growth;

[0028] (c) proliferation of lymphocytes at the base of the hairfollicle, a condition called lymphocytosis, poses a much seriousproblem—a condition called alopecia areata—in which there is excessivehair loss. This frequently occurs in women under stress, and causes astrong emotional response, as the hair comes off in clumps and iscosmetically disfiguring. Current treatment is to use a steroidcream—but it is of limited effectiveness and not quick in onset;

[0029] (d) proliferation of the epithelial cells of the sebaceous glandduring puberty and other conditions of excessive dihydrotestosteroneproduction contributes to the condition known as acne.

[0030] Doses and Deliveries

[0031] Typically a therapeutically effective dosage is formulated tocontain a concentration of at least about 0.1% w/w up to about 50% w/wor more, preferably more than 1% w/w of the active compound to thetreated tissue. The active ingredient may be administered at once, ormay be divided into a number of smaller doses to be administered atintervals of time or be administered as a sustained dose preparation.The term “sustained release formulation” is intended to encompassformulations that allow the continuous delivery of a CRH agonist to asubject over a period of time, preferably several days to weeks. Suchformulations are typically administered subcutaneously orintramuscularly and allow for the continual steady release of apredetermined amount of drug in the subject over time. Thesustained-release formulation of CRH agonist can be, for example, aformulation comprising a polymer selected from the group consisting of apoly-lactide polymer, a poly-glycolide polymer, and apoly-lactide/poly-glycolide copolymer (e.g., the drug is encapsulatedwithin a microcapsule comprising the polymer or copolymer). Suchsustained-release formulations, suitable for depot injection, are knownin the art for administration of peptide agonists, such as leuprolide(e.g. U.S. Pat. Nos. 4,677,191 and 4,728,721). The sustained-releaseformulation can be formulated to allow for delivery of the drug over apredetermined time period. Alternatively, the sustained-releaseformulation of CRH agonist may comprise a formulaic in an osmotic pump(i.e., the CRH formulation is enclosed with the osmotic pump). Suchosmotic pumps, which can be formulated to allow for release of apredetermined amount of drug over a predetermined time period, are knownin the art (e.g. the Alzet pump, commercially available from Alza, PaloAlto, Calif.). The dosage of CRH agonist released by thesustained-release formulation is preferably about 5-300microgram/kg/day, and more preferably 10-50 microgram/kg/day.

[0032] It is understood that the precise dosage and duration oftreatment is a function of the tissue being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed formulations.

[0033] The compound may be suspended in micronized or other suitableform or may be derivatized to produce a more soluble active product orto produce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the proliferativecondition and may be empirically determined.

[0034] Compounds are typically included at concentrations 0.001% w/w orgreater than 1% w/w up to 50% w/w or higher. The concentration isgenerally greater than the concentration for systemic administration ofthe compound. Preferable concentrations are in the range of 0.01% w/w toabout 25% w/w, more preferably 1% w/w to 25% w/w, yet more preferablygreater than about 1% w/w to about 10% w/w, and most preferably greaterthan 1% w/w up to about 5% w/w. Aqueous suspensions and formulationscontain 1% w/w or more.

[0035] Formulations

[0036] The suitable therapeutic formulations may be a solution,suspension, emulsion or the like, and may be formulated as creams, gels,ointments, emulsions, solutions, elixirs, lotions, suspensions,tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories,bandages, or any other formulations suitable for topical, intravenous,intradermal, or subcutaneous injection administration.

[0037] Pharmaceutical and cosmetic carriers or vehicles suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. In addition, the compounds may beformulated as the sole pharmaceutically active ingredient in thecomposition or may be combined with other active ingredients The activecompound is included in the carrier in an amount sufficient to exert atherapeutically useful effect in the absence of serious toxic effects onthe treated individual. The effective concentration may be determinedempirically by testing the compounds using in vitro and in vivo systems,including the animal models described herein.

[0038] For topical administration, the compounds may be formulated incompositions in the form of gels, creams, lotions, solids, solutions orsuspensions, or aerosols. Compositions for treating human skin areformulated for topical application with an anti-proliferative effectiveamount of one or more of the compounds selected as described herein, inan effective concentration range (by weight), between about 0.1% and80%, preferably 0.1 to 50%, more preferably greater than about 1% up toabout 50% or more in a cream, ointment, lotion, gel, solution or solidbase or vehicle known in the art to be nontoxic and dermatologicallyacceptable or suitable for application to the mucosa. Aqueoussuspensions are preferably formulated at concentrations greater thanabout 1% w/w, more preferably 2% w/w.

[0039] To formulate a composition, the weight fraction of compound isdissolved, suspended, dispersed or otherwise mixed in a selected vehicleat an effective concentration such that the proliferative condition isrelieved or ameliorated. Generally, emollient or lubricating vehiclesthat help hydrate the skin are more preferred than volatile vehicles,such as ethanol that dry the skin. Examples of suitable bases orvehicles for preparing compositions for use with human skin arepetrolatum, petrolatum plus volatile silicones, lanolin, cold cream, andhydrophilic ointment.

[0040] The choice of an acceptable vehicle is largely determined by themode of application and tissue to be treated. Suitable pharmaceuticallyand dermatologically acceptable vehicles for topical application includethose suited for use include lotions, creams, solutions, gels, tapes andthe like. Generally, the vehicle is either organic in nature or anaqueous emulsion and capable of having the selected compound orcompounds, which may be micronized, dispersed, suspended or dissolvedtherein. The vehicle may include pharmaceutically-acceptable emollients,skin penetration enhancers, coloring agents, fragrances, emulsifiers,thickening agents, and solvents.

[0041] Lotions

[0042] The lotions contain an effective concentration of one or more ofthe compounds. The effective concentration is preferably effective todeliver an anti-proliferative amount, typically at a concentration ofbetween about 0.1-50% w/w or more of one or more of the compoundsprovided herein. The lotions also contain from 1% to 50% w/w, preferablyfrom 3% to 15% w/w of an emollient and the balance water, a suitablebuffer, a C.sub.2 or C.sub.3 alcohol, or a mixture of water of thebuffer and the alcohol. Any emollients known to those of skill in theart as suitable for application to human skin may be used.

[0043] Creams

[0044] The creams are formulated to contain concentration effective todeliver an anti-proliferative effective amount of the compound to thetreated tissue, typically at between about 0.1%, preferably at greaterthan 1% up to and greater than 50%, preferably between about 3% and 50%,more preferably between about 5% and 15% of one or more of the compoundsprovided herein. The creams also contain from 5% to 50%, preferably from10% to 25%, of an emollient and the remainder is water or other suitablenon-toxic carrier, such as an isotonic buffer. The cream may alsocontain a suitable emulsifier. The emulsifier is included in thecomposition at a level from 3% to 50%, preferably from 5% to 20%.

[0045] Solutions and suspensions for topical administration areformulated to contain an amount of one or more compounds effective todeliver an anti-proliferative amount, typically at a concentration ofbetween about 0.1-50% w/w, preferably at least more than 1% w/w, morepreferably more than 2% w/w of one or more of the compounds providedherein. The balance is water, a suitable organic solvent or othersuitable solvent or buffer. Suitable organic materials useful as thesolvent or a part of a solvent system are as follows: propylene glycolpolyethylene glycol (M.W. 200-600), polypropylene glycol (M.W.425-2025), glycerine, sorbitol esters, 1,2,6-hexanetriol ethanol,isopropanol, diethyl tartrate, butanediol and mixtures thereof. Suchsolvent systems can also contain water.

[0046] Solutions or suspensions used for local application can includeany of the following components: a sterile diluent, such as water forinjection, saline solution, fixed oil, polyethylene glycol glycerine,propylene glycol or other synthetic solvent; antimicrobial agents, suchas benzyl alcohol and Methyl parabens; antioxidants, such as ascorbicacid and sodium bisulfite; chelating agents, such asethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,citrates and phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. Liquid preparations can be enclosed inampoules, disposable syringes or multiple dose vials made of glass,plastic or other suitable material Suitable carriers may includephysiological saline or phosphate buffered saline (PBS), and thesuspensions and solutions may contain thickening and solubilizingagents, such as glucose, polyethylene glycol, and polypropylene glycoland mixtures thereof. Liposomal suspensions, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art.

[0047] Suitably prepared solutions and suspension may also be topicallyapplied to the eyes and mucosa. Solutions, particularly those intendedfor ophthalmic use, may be formulated as 0.01%-10% w/w isotonicsolutions, pH about 5-7, with appropriate salts, and preferablycontaining one or more of the compounds herein at a concentration ofabout 0.1% w/w preferably greater than 1% w/w, up to 50% w/w or more.Suitable ophthalmic solutions are known (see, e.g. U.S. Pat. No.5,116,868, which describes typical compositions of ophthalmic irrigationsolutions and solutions for topical application). Such solutions, whichhave a pH adjusted to about 7.4, contain, for example, 90-100 mM sodiumchloride, 4-6 mM dibasic potassium phosphate, 4-6 AM dibasic sodiumphosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride,1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mMD.L.-sodium.beta.-hydroxy butyrate and 5-5.5 mM glucose.

[0048] Gels

[0049] Gel compositions can be formulated by simply admixing a suitablethickening agent to the previously described solution or suspensioncomposition. Examples of suitable thickening agents have been previouslydescribed with respect to the lotions.

[0050] The gelled compositions contain an effective amount of one ormore of an anti-proliferative amount, typically at a concentration ofbetween about 0.1-50% w/w or more of one or more of the compoundsprovided therein; from 5% to 75% w/w, preferably from 10% to 50% w/w, ofan organic solvent as previously described; from 0.5% to 20% w/w,preferably from 1% to 10% w/w of the thickening agent; the balance beingwater or other aqueous carrier.

[0051] Aspects of the invention will now be illustrated by the followingexamples, which are intended to illustrate but not to limit theinvention.

EXAMPLE 1

[0052] This example illustrates the anti-cancer property of an inventivepeptide with a D-amino acid as residue 20. Various tumor cells weretransplanted into mice or rats and the tumor volume was measured using acaliper-ruler. The volume of the tumor, in cubic mm, was estimated asthe long axis×the square of the short axis divided by 2.

[0053] In the experiment of Table 2, female mice, weighing 20-25 g, of aC57B1 line, were transplanted under the skin with B16 melanoma cells.After transplantation of tumors mice were mixed and divided into groupsof controls, CRH-treated and [D-Glu²⁰] CRH-treated. The test substancesat a dose of 0.1 mg/kg subcutaneously or the vehicle control (saline)was injected on days 3 to 7 after tumor transplantation, and the volumeof tumors were measured at various days afterwards. TABLE 2 Influence ofCRH and [D-Glu²⁰] CRH on Growth of B16 Melanoma in Mice Volume of Tumorsin mm³ DOSE Days after transplantation of tumors GROUPS (mg/kg) 10 12 1417 Controls — 59 + 9  221 + 38  296 + 62  382 + 118 hCRH 0.1 29 + 7*157 + 23* 133 + 15* 238 + 41  [D-Glu²⁰] CRH 0.1 37 + 7*  86 + 15* 138 +16* 252 + 54 

[0054] The second experiment and third experiment (data shown in Tables3 and 4) were conducted on white non-inbred male rats with asubcutaneous transplant of Walker carcinosarcoma or Ehrlich solidcarcinoma. Animals after transplantation of tumors were mixed and weredivided into groups of controls, CRH-treated and [D-Glu²⁰] CRH-treated.TABLE 3 Influence of CRH and [D-Glu²⁰] CRH on Growth of WalkerCarcinosarcoma in Rats DOSE (mg/kg), Volume of Tumors in mm³ N = no.Days after transplantation of tumors GROUPS of rats 7 10 12 14 Controls—, 8 345 ± 41 4288 ± 613 13193 ± 14096 ± 2361 1971 hCRH 0.1, 16 196 ±3600 ± 413 6942 ± 10832 ± 35*  905* 1146 [D-Glu²⁰] CRH 0.1, 16 127 ±3926 ± 633 6554 ± 10191 ± 22*  1041* 1260

[0055] TABLE 4 Influence of CRH and [D-Glu²⁰] CRH on Growth of EhrlichSolid Carcinoma in Rats DOSE (mg/kg), Volume of Tumors in mm³ N = no. ofDays after transplantation of tumors GROUPS rats 9 11 14 Controls  —, 8350 ± 13  776 ± 154 1265 ± 272  hCRH 0.1, 16 226 ± 14* 384 ± 61* 763 ±114* [D-Glu²⁰] CRH 0.1, 16 283 ± 33* 470 ± 80* 571 ± 199*

EXAMPLE 2

[0056] The peptide embodiment of the invention, [D-Glu²⁰] CRH, wasprepared where the normal 20 position glutamic acid of CRH had beensubstituted with D-glutamic acid. FIG. 1 graphically illustrates thehypotensive effects of unmodified CRH by contrast to the effect of[D-Glu²⁰]-CRH in anesthetized male rats. On the ordinate, the responseis given in units of integrated blood pressure observed over a 100minute period after the intravenous injection of the test substance ispentobarbital-anesthetized rats (no. of animals=8 per group per dose).The area-under-curve, is given in units of mm Hg per minute. Forexample, at a dose of 0.05 mg/kg peptide i.v., the response isapproximately 7200 mm Hg-min versus 1700 mm Hg-min for the D-amino acidsubstituted analog. That is, over a 100 minute period, the average fallin mean blood pressure is 72 mm Hg for CRH, but only 17 mm Hg for theD-amino acid substituted analog. This [D-Glu²⁰]-CRH analog embodiment ofthe invention provided much less hypotensive activity than that of theunmodified CRH (human/rat) control.

EXAMPLE 3

[0057] Example 3 illustrates an assay for the anti-proliferativeproperty of the D-amino acid residue 20 substituted peptide for thisinvention. We have further tested embodiments for theseanti-proliferative actions on human keratinocytes.

[0058] Methods and Materials

[0059] Human melanoma cells were maintained as monolayers in T25 cultureflasks (Corning) in Ham's F-10 media with 10% fetal bovine serum, 10μg/ml insulin, 250 U/ml penicillin G (Sigma), 50 U/ml polymyxin Bsulfate (Sigma), 100 μg/ml streptomycin sulfate (Pfizer), 2.5 μg/mlamphotericin B, and 10 μg/ml gentamicin solution. Cloudman S91 melanomacells had an additional 7% fetal horse serum added to the aboveformulation for optimal growth. Cells were incubated at 37° C. in ahumid atmosphere of 5% CO₂ and 95% air.

[0060] For generation of growth curves, cells were seeded in 96-welltissue culture plates (Dynatech) at 2000 cells/well with the abovemedia, except that 5% fetal bovine serum was used instead of 10%. Cellswere allowed to attach overnight and 24 hours after seeding, a baselinereading of cells were taken using the Aqueous Cell Proliferation AssayKit (Promega) based on the colorimetric assay method by Mosmann (MosmannT: Rapid calorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assay. J. ImmunologicalMethods, 65, pp. 55-66, 1983.). Ham's F-10 media with the abovesupplements with or without the [D-Glu²⁰] corticotropin releasinghormone (CRH) at the following concentrations: 10⁻⁶, 10⁻⁸ and 10⁻¹⁰ Mfor the duration of cell growth measurement. Cell growth was measuredevery 3-4 days using the Promega kit, absorbance measured at 492 nm on aTitertek Multiscan spectrophotometer, until cell death or maximum growthhad been achieved. Plates were maintained in a 37° C. incubator also ina humid atmosphere of 5% CO₂ and 95% air. [D-Glu²⁰], CRH, at lowconcentrations was able to inhibit melanoma cell growth in vitro.

[0061] Keratinocytes Proliferation

[0062] Two protocols were applied. The cells were seeded into 24 wellplates at the concentration of 50,000 cells/well in 0.5 ml of DMEM plus5% FBS. Next day the media were changed to DMEM containing 5% fetalbovine serum (final volume of 200 μl) supplemented with ³H [methyl-³H]thymidine (1 μCi/ml) and different concentrations of the [D-Glu²⁰] CRH.The cells were incubated for 6 hours, then the media were discarded, thewells washed twice with DMEM (1 ml), and the cells dissolved in 300 μlof 1 N NaOH. The resulting solution was mixed with liquid scintillationfluid and the radioactivity counted in the Beckman liquid scintillationspectrometer. Significant inhibition of DNA synthesis was noted at theconcentration 1 nM of [D-Glu²⁰] CRH. The data is illustrated by FIG. 2.

[0063] The cells were seeded into 24 well plates at the concentration of20,000 cells/well in 0.5 ml of DMEM plus 5% FBS. After 8 hours the mediawere changed and different concentrations of [D-Glu²⁰]CRH were added.After 24 hours the media were changed again and supplemented withdifferent concentrations of [D-Glu²⁰]CRH as well as with ³H [methyl-³H]thymidine (1 μCi/ml). The cells were incubated overnight, then the mediawere discarded, the wells washed twice with DMEM (1 ml), and the cellsdissolved in 300 μl of 1 N NaOH. The resulting solution was mixed withliquid scintillation fluid and the radioactivity counted in the Beckmanliquid scintillation spectrometer.

[0064] As shown by FIG. 2, significant and dose-dependent inhibition ofcell proliferation as measured by DNA synthesis, was noted at theconcentration as low as 10 pM of the ligand. The experiment was repeatedwith similar results.

[0065] It is to be understood that while the invention has beendescribed above in conjunction with preferred specific embodiments, thedescription and examples are intended to illustrate and not limit thescope of the invention, which is defined by the scope of the appendedclaims.

1 12 1 41 PRT Human/rat 1 Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu ThrPhe His Leu Leu Arg 1 5 10 15 Glu Val Leu Glu Met Ala Arg Ala Glu GlnLeu Ala Gln Gln Ala His 20 25 30 Ser Asn Arg Lys Leu Met Glu Ile Ile 3540 2 41 PRT Pig 2 Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe HisLeu Leu Arg 1 5 10 15 Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu AlaGln Gln Ala His 20 25 30 Ser Asn Arg Lys Leu Met Glu Asn Phe 35 40 3 41PRT Sucker fish 3 Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe HisLeu Leu Arg 1 5 10 15 Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu AlaGln Gln Ala His 20 25 30 Ser Asn Arg Lys Met Met Glu Ile Phe 35 40 4 41PRT Sheep/goat 4 Ser Gln Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His LeuLeu Arg 1 5 10 15 Glu Val Leu Glu Met Thr Lys Ala Asp Gln Leu Ala GlnGln Ala His 20 25 30 Ser Asn Arg Lys Leu Leu Asp Ile Ala 35 40 5 41 PRTCow 5 Ser Gln Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg 15 10 15 Glu Val Leu Glu Met Thr Lys Ala Asp Gln Leu Ala Gln Gln Ala His20 25 30 Asn Asn Arg Lys Leu Leu Asp Ile Ala 35 40 6 41 PRT Sucker fish6 Asn Asp Asp Pro Pro Ile Ser Ile Asp Leu Thr Phe His Leu Leu Arg 1 5 1015 Asn Met Ile Glu Met Ala Arg Ile Glu Asn Glu Arg Glu Gln Ala Gly 20 2530 Leu Asn Arg Lys Tyr Leu Asp Glu Val 35 40 7 41 PRT Carp 7 Asn Asp AspPro Pro Ile Ser Ile Asp Leu Thr Phe His Leu Leu Arg 1 5 10 15 Asn MetIle Glu Met Ala Arg Ile Glu Asn Glu Arg Glu Gln Ala Gly 20 25 30 Leu AsnArg Lys Tyr Leu Asp Glu Val 35 40 8 41 PRT Maggy sole 8 Ser Glu Glu ProPro Met Ser Ile Asp Leu Thr Phe His Met Leu Arg 1 5 10 15 Asn Met IleHis Arg Ala Lys Met Glu Gly Glu Arg Glu Gln Ala Leu 20 25 30 Ile Asn ArgAsn Leu Leu Asp Glu Val 35 40 9 41 PRT European flounder 9 Ser Glu AspPro Pro Met Ser Ile Asp Leu Thr Phe His Met Leu Arg 1 5 10 15 Asn MetIle His Met Ala Lys Met Glu Gly Glu Arg Glu Gln Ala Gln 20 25 30 Ile AsnArg Asn Leu Leu Asp Glu Val 35 40 10 40 PRT Rat 10 Asp Asp Pro Pro LeuSer Ile Asp Leu Thr Phe His Leu Leu Arg Thr 1 5 10 15 Leu Leu Glu LeuAla Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln 20 25 30 Asn Arg Ile IlePhe Asp Ser Val 35 40 11 40 PRT Human 11 Asp Asn Pro Ser Leu Ser Ile AspLeu Thr Phe His Leu Leu Arg Thr 1 5 10 15 Leu Leu Glu Leu Ala Arg ThrGln Ser Gln Arg Glu Arg Ala Glu Gln 20 25 30 Asn Arg Ile Ile Phe Asp SerVal 35 40 12 40 PRT Frog SITE (1) Xaa in position 1 is pyroglutamyl 12Xaa Gly Pro Pro Ile Ser Ile Asp Leu Ser Leu Glu Leu Leu Arg Lys 1 5 1015 Met Ile Glu Ile Glu Lys Gln Glu Lys Glu Lys Gln Gln Ala Ala Asn 20 2530 Asn Arg Leu Leu Leu Asp Thr Ile 35 40

We claim:
 1. A method of inhibiting cancerous tumor growth in mammals,comprising: administering a member of the corticotropin-releasinghormone (CRH) superfamily or analog thereof, wherein the 20th amino acidfrom the N-terminus of a 41 amino acid peptide or the 19th amino acidfrom the N-terminal of a 40 amino acid peptide of the CRH superfamily oranalog thereof is replaced with a D-amino acid peptide, wherein saidsuperfamily peptide or analog thereof has minimal activity to lowerblood pressure and the molecule retains anti-proliferative action. 2.The method as in claim 1, wherein the CRH peptide comprises the aminoacid sequence of SEQ ID NO:
 1. 3. The method as in claim 1, wherein theCRH superfamily peptide or analog thereof comprisesCyclo(30-33)-Acetyl-Pro⁴, D-Phe¹², D-Glu²⁰, Nle^(21,38), Glu³⁰, D-His³²,Lys³³-hCRH(4-41) or Cyclo(30-33)-Acetyl-Pro⁴, D-Phe¹², D-Glu²⁰,Nle^(28,21), Glu³⁰, D-Ala³², Lys³³-carp urotensin (4-41).
 4. The methodas in claim 1, wherein the cancerous tumor growth is a carcinoma,carcinosarcoma, melanoma or sarcoma.
 5. The method as in claim 1,wherein the cancerous tumor growth is bladder, brain, breast, colon,lung, prostate or skin cancer.
 6. The method as in claim 1, wherein thecancer is of bone, cartilage, connective tissue or striated muscle. 7.The method as in claim 1, wherein the administration comprises topical,systemic or parenteral administration.
 8. The method as in claim 7,wherein the administration is to the skin or mucous membrane of ananimal diagnosed as being at risk of a melanoma.
 9. The method as inclaim 1, wherein the CRH superfamily peptide or analog thereof compriseshuman/rat [D-Glu²⁰] CRH.
 10. The method as in claim 1, wherein theadministering is by intravenous, intradermal, subcutaneous, intranasalor intrabuccal means.
 11. The method as in claim 1, wherein the CRHsuperfamily peptide or analog thereof is formulated forsustained-release.
 12. The method of claim 1, wherein the the CRHsuperfamily peptide or analog thereof comprises a cyclo(30-33)-hCRH. 13.The method of claim 1, wherein the the CRH superfamily peptide or analogthereof comprises a [D-Glu²⁰]-cyclo(30-33)-hCRH.